TITLE: ?Coupling promoter choice and alternative RNA splicing in the mammalian Protocadherin gene cluster? PROJECT SUMMARY: Eukaryotic gene expression is regulated by a complex network of functional coupling between the processes of transcription initiation, elongation and RNA processing. The mammalian Protocadherin (Pcdh) gene cluster provides a remarkable and fundamentally important system to study the underlying mechanisms of this coupling process. The proteins encoded by the Pcdh gene cluster play an essential role in neural circuit assembly by providing individual neurons with a unique cell surface ?code? that forms the basis of self-recognition. The Pcdh cell surface code is generated by a complex process of stochastic promoter choice, alternative RNA splicing and combinatorial assembly of Pcdh cis-dimers at the cell surface. The transcriptional process involves ?stochastic? activation of individual Pcdh gene promoters through long-range enhancer-promoter DNA looping (which requires the DNA binding protein CTCF), transcription of as much as 250,000 base pairs of DNA, followed by splicing of a promoter proximal 5' splice site to a distant 3' splice site. Although significant progress has been made in understanding the genomic DNA organization, single neuron expression, and chromosome domain configuration of the clustered Pcdh, the mechanisms by which transcriptional initiation and elongation, and RNA processing are coupled remain unknown. Recent studies have revealed a remarkable organization of highly conserved RNA duplex structures in the Pcdh pre-mRNAs, and a striking pattern of convergent transcription at Pcdh promotors. These preliminary observations have lead to a model in which these RNA secondary structures regulate 5' splice site choice, and a novel mechanism for promoter choice. Aim 1 of this proposal is to Determine the architecture of Pcdh ? and ? RNA precursors in mammalian cells and investigate their functions, and Aim 2 is to Determine the role of CTCF in regulating transcription and processing of Pcdh? RNAs. A variety of approaches will be used to accomplish these aims, including the in vivo analysis of RNA secondary structures using chemical probes and RNASeq methods, single molecule visualization methods to image the translocation of RNA polymerase as it proceeds through the gene cluster, and gene editing methods to identify regulatory elements required for transcription and splicing. The proposed studies are poised to reveal novel and exciting regulatory mechanisms governing eukaryotic gene regulation. Moreover, as Pcdh proteins play a central role in neural circuit assembly, and they have been implicated in neurological diseases, understanding the details of Pcdh gene expression will not only provide fundamental insights into novel mechanisms of gene expression, but also lead to a better understanding of the genetic basis of neurological diseases, such as autism. Thus, the proposed research is of direct relevance to human health.